PROJECT SUMMARY/ABSTRACT Human cells integrate external and internal signaling inputs to make regulatory decisions that change functional outputs. Two such decisions of cells ? the decision to polarize and migrate and the decision to enter the proliferative state - are central to multicellular development and tissue maintenance. The regulatory feedbacks and the core switch mechanisms of how cells start to migrate or proliferate are not yet understood. Due to significant cell-to-cell variability and lack of synchronization, an understanding of the underlying regulatory motifs cannot be achieved by biochemical and genetic approaches alone. However, recently developed activity reporters and rapid perturbation strategies have made it possible to investigate complex spatial and temporal signaling feedback architectures and decision processes in living single cells, an approach that can reveal feedback mechanisms and circumvent the technical bulk assay issues. Our work seeks to understand the principles of cellular decision processes in human cells by employing these live-cell methods to monitor, perturb and automatically analyze the relevant signaling processes and ultimately derive quantitative models of how specific decisions are made. Our proposed work has Two Themes: In our first theme, we determine how cells initiate and establish cell polarity and how already polarized cells steer their front during directed migration and chemotaxis. We have developed automated fluorescence microscopy methods to monitor and perturb the critical Rho family small GTPases and relevant second messengers, and developed methods to quantify changes in different actin structures. These approaches will allow us to understand the core regulatory mechanisms for cell polarization and cell steering during migration. In our second theme, we seek to understand how cells decide to transition from a quiescent to a proliferative state by investigating molecular mechanisms of competition between stress and mitogens, by determining the molecular mechanism of the point-of-no return for cell cycle entry, and by exploring how sequential signaling events prevent the re-replication of the same DNA to ensure that DNA is only replicated once. We have developed a number of live cell cycle activity reporters, perturbation strategies and automated single-cell analysis methods that will help us understand and model the regulatory mechanisms controlling human cell cycle entry. Both our themes will lead to new concepts of the logics of human decision processes and will provide detailed molecular and mechanistic models explaining how cells integrate signaling inputs to start to migrate or enter the cell cycle. Finally, the universal dysregulation of cell proliferation and migration in cancer, and the frequent dysregulation of these processes in degenerative, immune and other diseases, argues that a molecular understanding of the complex regulatory architecture of cell migration and proliferation may lead to new therapeutic strategies for the treatment of a broad range of diseases.